Abstract

The Drosophila porcupine gene is required for secretion of wingless and other Wnt proteins, and sporadic mutations in its unique human ortholog, PORCN, cause a pleiotropic X-linked dominant disorder, focal dermal hypoplasia (FDH, also known as Goltz syndrome). We generated a conditional allele of the X-linked mouse Porcn gene and analyzed its requirement in Wnt signaling and embryonic development. We find that Porcn-deficient cells exhibit a cell-autonomous defect in Wnt ligand secretion but remain responsive to exogenous Wnts. Consistent with the female-specific inheritance pattern of FDH, Porcn hemizygous male embryos arrest during early embryogenesis and fail to generate mesoderm, a phenotype previously associated with loss of Wnt activity. Heterozygous Porcn mutant females exhibit a spectrum of limb, skin, and body patterning abnormalities resembling those observed in human patients with FDH. Many of these defects are recapitulated by ectoderm-specific deletion of Porcn, substantiating a long-standing hypothesis regarding the etiology of human FDH and extending previous studies that have focused on downstream elements of Wnt signaling, such as β-catenin. Conditional deletion of Porcn thus provides an experimental model of FDH, as well as a valuable tool to probe Wnt ligand function in vivo.

Generating and characterizing a conditional mouse Porcn allele. (A) Schematic diagram of WT, loxP-targeted, and deletion alleles of Porcn. Exons are boxed and numbered, with the coding region indicated in black. (B) Western blot of whole-cell lysates from parental and targeted ES cells (Left) and HeLa cells transfected with empty vector or mouse Porcn (Right) using a polyclonal antiserum against a C-terminal Porcn epitope. The diamond indicates the band corresponding to overexpressed mouse Porcn and is present only in parental and Porcnlox ES cells. (C and D) Whole-mount in situ hybridization for Brachyury (purple) on E6.5 control or PorcnΔ/Y embryos. (Scale bar: 100 μm.) (E and F) Sections through Brachyury whole-mount stained embryos, with approximate positions indicated by the dotted lines in C and D. Note that sections were taken from an independent representative pair of embryos, overstained to preserve signal in sections (asterisks indicate nonspecific background). PorcnΔ/Y embryos consistently contain a hollow lumen at this stage, indicating a lack of gastrulation.